CTCF-dependent expression of DUSP2.
a. HEK293 cells were transfected on two consecutive days with a pool of five different siRNAs against hCTCF (siCTCF) or a control siRNA (si ctrl). After 96 h the RNA was isolated and expression of CTCF and DUSP2 was analyzed by RT-PCR and normalized to ACTB (si control = 1). CTCF knockdown was performed 2 times and the qRT-PCR was done in triplicates and significance is indicated (same procedure for C). b. Reduction of CTCF protein after RNA interference was analyzed by western blot. GAPDH expression was utilized as control. c. Reduction of DUSP2 expression after siCTCF transfection in HeLa and HTB171 cells. d. Different CTCF constructs; wildtype, mutated SUMO site K > R at position 74 (CTCF-K > R74) and 691 (CTCF-K > R691), deletion of CTCF PARylation site (CTCF-ΔPAR) and vector control (pEGFP) were transfected in HEK293. After two days DUSP2 expression was analyzed by RT-PCR and normalized to ACTB (normal control = 1). Triplicates were determined and the data of three independent experiment were averaged and significance was calculated (same procedure for e and f) e. Expression of DUSP2 after transfection of different CTCF constructs in H322 cells (for details see d) f. DUSP2 expression was analyzed in CTCF-inducible TREx293 cells, a stable HEK293 cell line (CTCF TREx293) that allows inducible CTCF expression by tetracycline (5 μg/ml). After two days expression of DUSP2 was analyzed by RT-PCR and normalized to ACTB and compared to the uninduced control (unind. = 1). g. Expression of CTCF in TREx293 cells was analyzed by western blot